Several approaches to improve the efficiency of gene transfer into reconstituting hematopoietic stem cells are presently being evaluated by the Simian Gene Transfer and Bone Marrow Transplantation Program of the National Heart, Lung, and Blood Institute. These involve methods for 1) expansion of populations of cycling progenitors and stem cells in vitro and in vivo through the use of cytokines and chemotherapeutic agents; and 2) isolation of cellular fractions having a high proliferative capacity from bone marrow, peripheral blood, and neonatal cord blood. Over the past year the supernatant from a cell line that has been engineered to produce a high titer of a replication-defective retrovirus that contains the human glucocerebrosidase gene has been used in autologous bone marrow transplants using cells that were immunoselected for CD34, and Thy-1 expression. CD34 is a transmembrane glycophosphoprotein that appears to be the L-selectin ligand and is expressed on a population of early hematopoietic cells which contains the reconstituting hematopoietic stem cell. Thy-1 is a transmembrane protein of unknown function that may be involved in cell activation and stromal adherence. Cells of the CD34+, Thy-1+ immunophenotype have been shown to have the capacity to initiate long-term bone marrow cultures, and may contain the repopulating hematopoietic stem cell. Immunoselected CD34+Thy-1+ cells have been cultured for 6 days in medium conditioned by the retrovirus producer cell line and combinations of growth factors. Following total body gamma-irradiation (TBI) (6.5yx2), the transduced cells have been reinfused, and peripheral blood cells analyzed at regular intervals for evidence of gene transfer using a polymerase chain reaction )PCR) asay. 6-10% of the circulating leukocytes have contained the human glucocerebrosidase gene. Presently, the program is attempting to use other vectors, such as the adeno-associated virus, in an attempt to improve upon the gene transfer efficiency observed in longterm reconstituting stem cells. With continued improvements in vector design, and continued advancements in the understanding of stem cell biology and gene regulation, studies utilizing primate models will prove of major importance in developing suitable protocols permitting safe and effective gene transfer into human subjects.